The field of the present invention pertains to a dry solid medium and method for collection of genetic material in a form suitable for storage or subsequent analysis. The invention further provides for analysis of stored genetic material including methods suited for automated analyzing systems.
Blood containing genetic material (including DNA or RNA) to be analyzed has typically been transported from the place of removal, from a human or animal, to the place of analysis as purified genetic material, liquid whole blood, frozen whole blood or whole blood dried onto paper. However, all of these methods have disadvantages. Transport of genetic material in blood as dried, purified genetic material is most desirable, but it requires a high standard of technical assistance to be available at the place of removal from the human or animal. When technical assistance is not available at the place of removal, whole blood or other unpurified samples are usually sent to a central facility where the genetic material is-purified.
Transport of liquid whole blood often involves the need for sterility of collection. Under some circumstances, this is extremely inconvenient, for example, where the sample is a heel-prick taken from an infant. The transport of liquid whole blood or frozen blood may also require temperature controlled transport or a transport system other than the regular postal system. This is true even before considering public health concerns. In addition, concern over pathogens associated with whole blood, such as the HIV virus, generally rule out the transport of a potentially infectious liquid or frozen sample except under proper and expensive supervision.
Transport of blood dried on filter paper is a proven alternative to the above procedures. Genetic material can be extracted and isolated from whole blood spots which are dried on filter paper, in a form and in sufficient quantities for use in genetic analysis. McCabe, E. R. B., et al., xe2x80x9cDNA Microextraction From Blood Spots on Filter Paper Blotters: Potential Screening Applications to Newborn Screening,xe2x80x9d Hum. Genet. 75:213-216 (1987). However, the procedure still suffers from a number of disadvantages. For example, typically, there has been no deliberate and rapid destruction of blood associated microorganisms. The presence of pathogenic microorganisms may create a potential hazard for blood handling personnel. Moreover, some microorganisms may cause damage to the genetic material. While some inhibition of microorganisms may occur through desiccation, it is known that slow desiccation, or even a small degree of rehydration under conditions of high relative humidity, may allow the growth of DNA or RNA destroying microflora.
Another disadvantage of present methods for dry transport of genetic material in blood is a lack of deliberate inhibition of processes which degrade genetic material. Hence, even in the presence of a bacteriostatic agent there are conditions that permit enzymatic, nonenzymatic and autolytic breakdown of the genetic material. Furthermore, using presently available methods of dry storage, considerable difficulty is encountered if desorption of high molecular weight DNA or RNA is required. The surface adsorption affects can cause loss of genetic material which may cause the preferential loss of the least degraded, i.e., the most desired class of DNA or RNA molecules.
Thus, there is a need for a safe, convenient and minimally labor intensive means for storage of a genetic material which is contained in a liquid sample.
However, even if a sample of genetic material is collected in a safe, convenient and reliable form for storage, subsequent analysis of the stored sample may give rise to logistic problems. This is especially true when there are many different types of analyses to be performed on a collected sample. The logistics tend to become even more complex when multiple samples are submitted for analysis.
For example, some methods of analysis of genetic material for a specific genetic sequence, such as polymerase chain reaction (PCR) analysis, require use of an oligonucleotide primer-pair. Generally a different primer-pair is used for each sequence analysis to be performed. And, because there are an extremely large number of possible primer pairs (for example, one for each sequence within the genes of humans, animals and all other living organisms including the pathogens of humans and animals), multiple sequence analyses in a single sample may give rise to immense logistic problems. The problems are potentially greater when multiple samples of genetic material are received for analysis, such as at a centralized analyzing facility.
Automated analysis of genetic material provides enhanced efficiency for processing large numbers of samples. However, automated analysis of a sample of genetic material still requires the automated system to have separate delivery devices for each different set of primers to prevent the occurrence of cross-contamination. Therefore, the range of genetic sequence analyses which can be performed at one time may be restricted due to the inherent problem of cross contamination of primers. Hence, there is a need for reducing the restrictions against analyzing multiple sequences or multiple samples of genetic material using automated systems.
The present invention provides a safe, convenient and minimally labor intensive apparatus and method for storage or analysis of a genetic material contained in a liquid medium. The invention further provides for storage of genetic material in such a way as to allow for simplified analysis of one or more samples of genetic material using automated systems.
The invention includes a dry solid medium for storage of a sample of genetic material. The dry solid medium of the invention is composed of a solid matrix and a composition which when applied to the dry solid medium protects against degradation of genetic material stored on the dry solid medium. The dry solid medium may further provide inactivation of microorganisms, including those which may be pathogenic to humans.
The composition of the dry solid medium may include a weak base, a chelating agent and a protein denaturant such as a detergent. In another embodiment, if long-term storage of a sample of genetic material is desired, the composition may further include a free radical trap.
According to the invention, genetic material stored on the dry solid medium may be analyzed using methods known in the art, for example, polymerase chain reaction (PCR), ligase chain reaction (LCR), reverse transcriptase initiated PCR, DNA or RNA hybridization techniques including restriction fragment length polymorphism (RFLP) and other techniques using genetic or DNA or RNA probes, genomic sequencing, enzymic assays, affinity labeling, methods of detection using labels or antibodies and other similar methods.
Genetic material stored on the dry solid medium of the invention may be analyzed in situ or after removal from the dry solid medium.
In another embodiment of the invention, the dry solid medium of the invention may include a component which is functional in subsequent analysis of the stored sample of genetic material. According to this embodiment, a component for subsequent analysis includes, for example, nucleotide sequences such as PCR primers, target sequence stabilizers, genetic probes, primers for genetic sequencing or sets of oligonucleotide substrates for LCR analysis. As disclosed herein, the compound for subsequent analysis may also include a suitably stable enzyme.
The invention also provides methods for using the dry solid medium. In one embodiment, the invention provides for storing a sample of genetic material on the dry solid medium in a substantially nondegraded form. The invention further provides a method for storage and subsequent analysis of a stored sample of genetic material. In a preferred embodiment, prior to analysis, the stored genetic material may be washed to remove compounds which may be associated with a sample of genetic material and which may inhibit subsequent analysis. Such compounds include, for example, protein, hemoglobin and components of the composition of the dry solid medium. The invention provides for the stored genetic material to be washed using an aqueous or a nonaqueous wash system.
The aqueous or nonaqueous wash system used to wash a sample of genetic material preferably provide for removing compounds which may inhibit subsequent analysis, without substantially affecting the stored genetic material or a component for subsequent analysis which may be included on the dry solid medium. Moreover, the aqueous and nonaqueous wash systems of the invention may also be used to wash genetic material which is stored, for example, on a solid matrix which does not have sorbed to it the composition of the invention.
In one embodiment, the nonaqueous wash system of the invention provides for contacting a sample of genetic material stored on the dry solid medium of the invention with a herein described single phase phenol solution. The single phase phenol solution is removed and the dry solid medium containing a sample of genetic material is subsequently contacted with an aqueous alcohol wash solution. The aqueous alcohol wash solution is removed and the sample of genetic material may further be washed with an aqueous ionic solution. The washed sample of genetic material may then be analyzed by methods known in the art, including those mentioned above.
Aqueous wash systems are known in the art. Under some circumstances, a wash system may cause some loss of a component for subsequent analysis which may be included on the dry solid medium of the invention. In one embodiment of the invention, a herein disclosed retaining agent may be used to reduce loss of a component for subsequent analysis during processing of the sample of genetic material.
A sample of genetic material stored on a dry solid medium of the invention may also be analyzed using standard enzymic tests used, for example, to detect phenylketonuria (PKU) or galactosemia, preferably after neutralizing the effects of protein denaturing components of the dry solid medium using, for example, a herein disclosed converter solution.
The dry solid medium and the dry solid medium including a component for subsequent analysis are particularly useful for analysis of a sample of genetic material using automated systems.